Western Blot total loaded protein amounts per lane - (Jan/12/2007 )
Hi Im new to the forum and I hope someone out there might be able to help me. I am doing Western blots to probe some neural precursor cells for phospho p53 at serine 15 (cell signaling). I was wondering if anyone knows the least amount of protein you can load per lane to get a nice, relatively strong signal. I know anywhere around 20ug and above is preferred, however I cannot load more than 5ug per sample since my cells were unfortunately too diluted in my lysis buffer. I know that phospho proteins are already quite weak in signal and that more protein is better than less, but I would just like to know what is the bare minimum?
Also, Im looking for some new serine phosphatae inhibitors because I hear that the okadaic acid Im currently using is not very potent (IC50 very high) and I want to look at some other options.
Thanks in advance for any help! 
Hi,
...so as you have not more than 5µg of sample to load, what can you do other than load the 5µg? If you still have these cells you can also do the isolation again, or?
What might help to get a stronger signal with this small amount is to use ECL plus (if you use HRP-conjugated secondary antibody) and too increase exposure time for development of the blot.
If you don't know if p53 is phosphorylated anyway in your cells I would do another staining which works; e.g. actin or tubulin so you can see how strong your signal is of these proteins with 5µg.
Good luck,
Cheers
Also, Im looking for some new serine phosphatae inhibitors because I hear that the okadaic acid Im currently using is not very potent (IC50 very high) and I want to look at some other options.
Thanks in advance for any help!
lot of questions; to get more answers to all your questions, better split up for distinct posts;
the good message is that normally anti-phospho protein Ab´s give stronger signals than the anti non-phospho protein Ab´s; better do an overnight exposure to optimize binding; there are forum discussion how weak immunosignals can be increased but may be it is not necessary, just start...
as I know okadaic acid has an IC50 for PP2A of 0.5 - 1 nm, that is excellent; for PP1 its ca. 200 nm, I think that is what you mean; to block both PP1 and PP2A, use microcystin LR (IC50 for both 0.1 nm) or calyculin A (2 nm for PP1, 0.5-1 nm for PP2A), all are not cheap but to prefer to NaF, naphtylphosphate or other unspecific stuff
increasing
make a bigger gel with larger wells and load more volume
try with 5 µg.
If it doesn't work, you can concentrate your sample by performing a DOC/TCA precipitation.
add 1/100 volume of deoxycholate 2%, vortex, let sit 30 minutes on ice.
add 1/10 volume of Trichloracetic acid 100%, vortex, let sit overnight at 4°C.
centrifuge 15000g 20 minutes.
optionnaly wash with cold acetone twice.
resuspend the pellet with laemmli buffer.
so you are able to load more.